GLUT4, the insulin responsive-glucose transporter, mediates the rate limiting step of glucose metabolism in skeletal muscles and adipose tissues. Exercise training up-regulates and High-fat feeding down-regulates GLUT4 mRNA. By using 5'-deleted mouse GLUT4 minigene transgenic mice, we have previously demonstrated that exercise and high-fat responsive elements of mouse GLUT4 are located 558 bp between -1000 and -442 relative to transcription initiation. To narrowing this area, -701, -551 and -423 GLUT4 minigene transgenic mice were made and the effects of exercise and high fat responsive elements are located -551〜-442 and -701〜-551. Furthermore, denervation responsive element is located downstream of -423 and different to exercise responsive element.Gel shift analysis was performed to test the binding activities of muscle nuclear extracts to GLUT4-1:-556〜-527, GLUT4-2:-531〜-502, GLUT4-3:-506〜-470, GLUT4-4:-476〜-438. The binding to GLUT4-3 was greater in nuclear extracts from exercised m
… Moreice than those of non-exercised mice. Moreover, DNase I footprinting analysis using -751〜-552 revealed that WAT nuclear extracts from high-carbohydrate and high-fat fed mice protected the region from -706 to -683 and from -674 to -652 from DNase I digestion. Therefore, gel shift analysis was performed to test the binding activities of WAT nuclear extracts from both diet fed mice using WF1:-712〜-688, WF2:-690〜-650, WF3:-655〜-616, WF4:-620〜-581, WF5:-585〜-546. The binding to WF4 and WF5 was more decreased in mice fed the high-fat diet than those fed the high-carbohydrate diet. These results demonstrated that increased binding factor to GLUT4-3 and decreased binding factor to WF4, WF5 may be responsible for up- and down-regulation of GLUT4 by exercise and high-fat feeding, respectively.次に、-551〜-442の領域内で、GLUT4-1:-556〜-527、GLUT4-2:-531〜-502、GLUT4-3:-506〜-470、GLUT4-4:-476〜-438のフラグメントを作成し、ゲルシフトアッセイを行った結果、GLUT4-3にスイミング運動により結合の増加する因子が存在した。また、-701〜-551の領域を含むフラグメントF1:-751〜-552を作成し、フットプリントを行った結果、-706〜-683と-674〜-652に蛋白質の結合領域が認められた。そこで、WF1:712〜-688、WF2:-690〜-650、WF3:-656〜-617、WF4:-621〜-582、WF5:-586〜-547のフラグメントを用いてゲルシフトアッセイを行ったところ、WF4とWF5に高脂肪食によって結合の減少する因子が存在した。これらの結果より、-506〜-470(GLUT4-3)、-621〜-582(WF4)、-586〜-547(WF5)に結合する因子が、GLUT4の運動による発現増加、高脂肪食による発現減少に関与している可能性が示唆された。 Less